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PubMed2003

Identification and enrichment of spermatogonial stem cells displaying side-population phenotype in immature mouse testis.

In mammals, spermatogenesis is maintained by spermatogonial stem cells (SSC). In their niche, SSC divide to self-maintain and to produce a transit-amplifying population that eventually enters the meiotic cycle to give rise to spermatozoa. The low number of SSC and the lack of specific markers hinder their isolation and enrichment. Stem cells in several adult tissues can be identified by using their verapamil-sensitive Hoechst dye-effluxing properties, which define the characteristic "side population" (SP). Here we show, by multicolor flow cytometric analysis, that immature mouse testis contains a "side-population" (T-SP), which is Sca-1pos, Ep-CAMpos, EE2 pos, alpha6-integrin pos, and alpha(v)-integrin neg. A 13-fold enrichment in SSC activity was observed when sorted T-SP cells from ROSA 26 mice were transplanted in busulfan-treated mouse testis. Whereas an incomplete range of spermatogenic stages was encountered two months after transplantation of unsorted testicular cells, the transplantation of T-SP cells generated all associations of mouse germ cells representing the full range of spermatogenic stages. These data suggest that Hoechst staining and cell sorting might provide a novel approach to SSC enrichment in mammals.

Animals↗
PubMed2004

Identification of the spermatogenic zip protein Spz1 as a putative protein phosphatase-1 (PP1) regulatory protein that specifically binds the PP1cgamma2 splice variant in mouse testis.

The spermatogenic zip protein (Spz1) was originally isolated from a mouse testis library and identified as a novel member of the basic helix-loop-helix family of transcription factors. Here we identify Spz1 as a specific binding partner of the gamma2 catalytic subunit of protein phosphatase-1. Male mice homozygous for a null mutation in the protein phosphatase-1cgamma (PP1cgamma) gene are infertile and display a distinct impairment in spermiogenesis despite the continued presence of closely related PP1c isoforms. Yeast two-hybrid screening using the PP1cgamma2 splice variant has identified Spz1 as an interacting protein and possible mediator of the sterile PP1cgamma mutant phenotype. Spz1 was shown to interact specifically with PP1cgamma2 but did not show an interaction with PP1calpha or with a truncated version of PP1cgamma2 lacking 18 amino acids from the C terminus. Interaction between full-length Spz1 and PP1cgamma2 was verified by co-immunoprecipitation and co-localization experiments in COS-1 cells as well as gel-shift and sedimentation assays using whole testis lysates. Immunohistochemistry on wild type testis sections reveals a stage-specific expression pattern for Spz1 during spermatogenesis that appeared grossly abnormal in the testes of PP1cgamma mutant mice. Phosphatase assays using recombinant PP1c indicate that increasing concentrations of Spz1 are able to inhibit PP1cgamma2 activity while having little effect on the activity of PP1calpha. Furthermore, an interaction between PP1cgamma2 and Spz1 was shown to prevent binding of the latter to the consensus E-box promoter sequence. We propose that the interaction between Spz1 and PP1cgamma2 may be required for proper regulation of spermatogenesis and fertility in males.

Alternative Splicing↗
PubMed1985

Investigation of thermotolerance in mouse testis.

The effect of a two-fraction heat treatment on mouse testis has been assessed by measuring testis weight loss at 1 week after treatment. The rate of repair of 'sublethal' heat damage following the first treatment was dependent on the severity of the treatment. Using a primary treatment of 41.5 degrees C for 30 min, the weight loss following a test treatment of 41.5 degrees C for 30 min returned to that of the test treatment alone within an interval of 16-24 h. Using a milder primary treatment of 40.0 degrees C for 30 min, repair of sublethal heat damage appeared to be complete by 1-2 h. When a single test treatment was used, there was no evidence of heat-induced thermal resistance (thermotolerance) following primary treatments of 40.0 or 41.5 degrees C for 30 min, for periods up to 24 h between treatments. A small degree of thermotolerance could, however, be demonstrated following the most severe primary treatment used if full dose: effect curves were obtained. Thermotolerance, manifest as a decrease in slope, was maximal at approximately 4 h after the primary treatment. The results are discussed with reference to other normal tissue data.

Animals↗
PubMed1990

Immunocytochemical localization of the epidermal growth factor receptor in mouse testis.

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.

Animals↗
PubMed1994

Molecular cloning and expression of cDNAs encoding two isoforms of protein phosphatase 2C beta from mouse testis.

Two isoforms of protein serine/threonine phosphatase were isolated and sequenced from mouse testis. A deletion of 48 nucleotides of PP2C beta 4 cDNA in comparison with PP2C beta 3 cDNA resulted in different COOH-terminal sequences of 12 and 15 amino acids, respectively. These COOH-terminal sequences of PP2C beta 3 and PP2C beta 4 were further found to be different from those of isoforms MPP beta 1 and MPP beta 2 of mouse PP2C beta reported (Terasawa, et al. Arch. Biochem. Biophys. 307: 342-349, 1993). The common sequence of 378 amino acids from these four isoforms of mouse PP2C beta exhibited 95% identity with the corresponding sequence of rat PP2C beta. The mRNAs of approximately 2.0 Kb for PP2C beta 3 and PP2C beta 4 were expressed only in testis, while the mRNAs of 3.3 Kb and 8.5 Kb for MPP beta 1 and MPP beta 2, respectively, were found in somatic tissues.

Amino Acid Sequence↗
PubMed2004

Murine Asb-17 expression during mouse testis development and spermatogenesis.

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Localization and characterization of an orphan receptor, guanylyl cyclase-G, in mouse testis and sperm.

We recently identified a novel testis-enriched receptor guanylyl cyclase (GC) in the mouse, designated mGC-G. To further investigate its protein expression and function, we generated a neutralizing antibody specifically against the extracellular domain of this receptor. RT-PCR and immunohistochemical analyses show that mGC-G is predominantly expressed from round spermatids to spermatozoa in mouse testis at both the mRNA and protein levels. Flow cytometry and confocal immunofluorescence reveal that mGC-G is a cell surface protein restricted to the plasma membrane overlying the acrosome and midpiece of the flagellum in mature sperm. Interestingly, Western blot analysis demonstrates that testicular mGC-G is approximately 180 kDa but is subject to limited proteolysis during epididymal sperm transport, resulting in a smaller fragment tethered on the mature sperm surface. On Fluo-3 cytometrical analysis and computer-assisted sperm assay, we found that serum albumin-induced elevation of sperm intracellular Ca(2+) concentration, protein tyrosine phosphorylation, and progressive motility associated with capacitation are markedly reduced by preincubation of the anti-mGC-G neutralizing antibody. Together, these results indicate that mGC-G is proteolytically modified in mature sperm membrane and suggest that mGC-G-mediated signaling may play a critical role in gamete/reproductive biology.

Animals↗
PubMed1976

Induction of meiosis in fetal mouse testis in vitro.

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a "meiosis-preventing substance" (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a "meiosis-inducing substance" (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.

Animals↗
PubMed1997

Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene.

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain (DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, gHDHC7 and gMDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.

Amino Acid Sequence↗
PubMed1985

Biochemical and immunological characterization of an H2A variant from the mouse testis.

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.

Amino Acids↗
PubMed2002

Expression and localization of prolyl oligopeptidase in mouse testis and its possible involvement in sperm motility.

Prolyl oligopeptidase (POP) expression in mouse testis during sexual maturation was examined. Northern blot analysis showed that POP mRNA expression was highest at 2 weeks of age, and gradually reduced thereafter. However, enzyme activity was almost constant during the examined period. In situ hybridization study revealed a change in the expression site of POP mRNA in testis during sexual maturation. Positive signals were detected in all types of cells in the seminiferous tubules before maturation, and were restricted to spermatids at the spermatogenesis cycle stages I-VIII in adult mice. POP was detected in the insoluble fraction of sperm by Western blot analysis. Immunohistochemical analyses showed that POP is localized in the spermatids at steps 12-16 of spermiogenesis and in the midpiece of the sperm fragellum. It was also found that specific POP inhibitors, poststatin and benzyloxycarbonyl-proline-prolinal, suppressed sperm motility. These results suggest that POP may be involved in meiosis of spermatocytes, differentiation of spermatids, and sperm motility in the mouse.

Aging↗
PubMed1997

Identification and expression of epidermal growth factor gene in mouse testis.

Epidermal growth factor (EGF) is produced primarily by Leydig cells of human testis. Expression of the EGF gene was assessed in mouse testis during the course of sexual maturation by the application of the RT-PCR method and the use of specific oligonucleotide primers. Testis EGF mRNA content increased with the developmental age of the mice, i.e., day 15 < day 30 < day 45 postnatal. The expression of the EGF gene appears to correlate with maturation of the testis and proliferation of Leydig-cells.

Animals↗
PubMed2001

Expression of prostaglandin D synthetase during development in the mouse testis.

Prostaglandin D synthetase is expressed relatively highly in the testis and reproductive tract of a number of species, including the mouse. In adult mouse testis, expression is confined largely to the Leydig cells and in this study changes in the expression and localization of prostaglandin D synthetase mRNA during testis development were examined. Initial studies using RT-PCR and isolated testicular compartments indicated that prostaglandin D synthetase expression in the neonatal testis was predominantly within the seminiferous tubules. In situ hybridization studies confirmed that prostaglandin D synthetase mRNA appears to be expressed only in the tubules of neonatal mouse testes and only in the interstitial tissue of the adult testis. TaqMan real-time PCR was used to quantify prostaglandin D synthetase mRNA content during development using an exogenous mRNA as a control standard. Expression per testis decreased after birth to < 10% at day 15 before recovering again by days 25-30. After day 30, expression per testis increased 40-fold during final development to adulthood. Studies using RT-PCR showed that early expression before day 15 was restricted to the tubular compartment, whereas the subsequent increase in expression after day 30 was restricted to the interstitial compartment. Database analysis showed that the 3' end of the prostaglandin D synthetase transcript was subject to alternate splicing. Both splice isoforms were shown by RT-PCR to be present throughout development and without a major change in expression pattern. These results indicate that expression of prostaglandin D synthetase mRNA shifts during development from the tubular compartment of the fetal or neonatal testis to the developing adult Leydig cells, with expression in the Leydig cells increasing markedly after puberty. These changes are similar to those observed for 17beta-hydroxysteroid dehydrogenase type III and may indicate that this developmental process is not uncommon in the testis.

Alternative Splicing↗
PubMed1984

Purification of non-histone acceptor proteins for ADP-ribose from mouse testis nuclei.

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.

Adenosine Diphosphate Ribose↗
PubMed1982

Damaging effects of fourteen chemotherapeutic drugs on mouse testis cells.

The harmful effects of 14 chemotherapeutic drugs on spermatogenesis in the mouse have been evaluated by studies of testicular cell killing and morphological and genetic damage produced. Male mice were given drugs as single injections at various doses up to the toxic levels. Prednisone and 6-mercaptopurine produced little or no cytotoxicity. All other drugs tested killed differentiated spermatogonia. Of these, methotrexate, cyclohexylchlorethylnitrosourea, cis-platinum, and mechlorethamine did not show significant stem cell killing. Bischlorethylnitrosourea, chlorambucil, 5-fluorouracil, mitomycin C, antinomycin D, and procarbazine showed some stem cell killing. Triethylenethiophosphoramide (thio-TEPA) was the only drug in this group which killed large numbers of stem cells. Only 5-fluorouracil and cis-platinum killed spermatocytes, and only cis-platinum killed spermatids. Several drugs induced chromosome breaks in treated spermatocytes. Thio-TEPA was effective in inducing chromosome translocations in treated spermatocytes and probably also in spermatocytes which originated from surviving treated stem cells. It had been our hypothesis that the cytotoxic effects of these drugs on mouse testicular stem cells would correlate with the duration of azoospermia observed in patients. This was shown not to be the case. Thus, the cytotoxic effects of single injections of single chemotherapeutic agents on the mouse testis did not appear to be predictive of which drugs will cause long-term azoospermia in humans.

Animals↗
PubMed1995

Ultracytochemical study of trimetaphosphatase activity during acrosomal formation in the mouse testis.

The localization of Trimetaphosphatase (TMPase) activity during the acrosomal formation in the mouse testis was enzyme cytochemically investigated by the cerium-salt method. In addition to the lysosomes of the Sertoli cells and the spermatogenic cells in the seminiferous tubules, positive TMPase activity was detected in the Golgi complex and in the acrosomal vesicles of the spermatids, as well as in the acrosomes of both spermatids and spermatozoa. In the Golgi complex of the spermatids, TMPase activity was observed in the first one or two lamellae of the trans-face and in the small vesicles in the vicinity of the Golgi complex. TMPase positive reaction was also detected in the acrosomes of the spermatozoa in the lumina of both the seminiferous tubules and the epididymal duct. The localization of this enzyme activity was compared with that of acid phosphatase (ACPase), as detected by the cerium-based method, using beta-glycerophosphate as substrate: ACPase activity was completely absent from the Golgi complex, small vesicles, acrosomal vesicle and acrosome throughout the entire process of acrosomal formation. TMPase is thought to become one of the acrosomal components, and may be involved in the acrosomal reaction during fertilization.

Acid Anhydride Hydrolases↗
PubMed2003

Effects of adenovirus mediated gene transfer to mouse testis in vivo on spermatogenesis and next generation.

PURPOSE: We directly injected DNA into mouse testes in vivo using an adenovirus vector to transfect testicular cells. We then analyzed the transfection efficiency, immunological problems and effects of gene transfer on spermatogenesis and the next generation. In this study we discuss the potential of gene therapy for male infertility. MATERIALS AND METHODS: A replication incompetent human adenovirus serotype 5 contained 2 deletions (E1 and E3 deletions) and was constructed such that the transgene was driven by the chicken beta-actin promoter to promote over expression of the downstream target gene (Lac Z). This adenovirus vector or control solution was injected into the interstitial space (intratesticular injection) or seminiferous tubules (intratubular injection) of the mouse testis. We investigated beta-galactosidase gene expression by X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining, the effects of gene transfer on spermatogenesis by evaluating the frequency of apoptotic cells by the TUNEL method, the inflammatory response on testes by detecting CD4 and CD8 positive cells immunohistochemically, and interleukin (IL)-6 and IL-8 by immunoblot analysis, epididymides sperm motility and the reproductive response of each mouse 3, 7, 14 and 28 days after injection. RESULTS: Intratesticular injection of adenovirus vector resulted in strong transgene expression in Leydig cells. In contrast, intratubular injection resulted in strong expression in Sertoli cells. Transgene expression was not detected in germ cells by either method. The peak of beta-galactosidase activity was on day 7, ie 0.674 +/- 0.20 (intratesticular) and 0.534 +/- 0.22 U (intratubular), and it decreased with time thereafter. The apoptosis index on day 7 was significantly higher in adenovirus injected groups than in noninjected groups, ie 0.46 +/- 0.20 vs 0.10 +/- 0.11 (intratesticular) and 0.78 +/- 0.31 vs 0.24 +/- 0.10 (intratubular). Transfected animals showed a slight mononuclear inflammatory response in the testes composed of CD4 and CD8 positive cells. Adenovirus vector stimulation resulted in the induction of IL-6 and IL-8 secretion in the testis. These immune responses subsided after day 7. There were no significant differences in the percent of motile sperm or the rate of abnormal sperm between the groups on any day after injection. Reproductive ability remained almost normal even after adenovirus mediated gene transfer with no effect observed in offspring. CONCLUSIONS: Our results suggest that although slight spermatogenic damage and inflammatory response caused by these methods may present problems, adenovirus mediated gene transfer may be effective for transfecting testicular somatic cells and applicable for in vivo gene therapy for male infertility in the future.

Adenoviruses, Human↗
PubMed1995

Regulation of cholesterol side-chain cleavage cytochrome P450 in mouse testis Leydig cell line I-10.

We have characterized regulation of steroidogenesis in a mouse testis Leydig cell line, I-10. Progesterone secretion was increased in a time- and dose-dependent fashion by 8-Br-cAMP treatment. The amount of cholesterol side-chain cleavage enzyme (P450scc), the first and rate-limiting enzyme for the synthesis of steroids, as detected by immunoblotting, was also increased. The calcium ionophore A23187 decreased the amount of P450scc, but it did not interfere with cAMP stimulation of the accumulation of P450scc. This regulation of P450scc expression by cAMP and A23187 is at the post-transcriptional level because the amount of P450scc mRNA was not affected by either treatment. This result was further confirmed by direct measurement of transcription in the presence or absence of forskolin treatment. I-10, however, supported cAMP-dependent transcriptional activation of the exogenous gene, as shown by the increased expression of a reporter gene under the control of the -600 to -2,500 fragment of the P450scc gene. The ability for transcriptional activation of the exogenous but not endogenous P450scc gene makes I-10 a unique steroidogenic cell line.

8-Bromo Cyclic Adenosine Monophosphate↗